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Soil Biology & Biochemistry 32 (2000) 1393±1403
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Identi®cation of tolerance to soil acidity in inoculant strains of
Rhizobium leguminosarum bv. trifolii
Elizabeth L.J. Watkin*, Graham W. O'Hara, John G. Howieson, Andrew R. Glenn 1
Centre For Rhizobium Studies, School of Biological Sciences and Biotechnology, Division of Science and Engineering, Murdoch University,
Murdoch, WA 6150, Australia
Received 14 September 1999; received in revised form 8 February 2000; accepted 23 February 2000

Abstract
The acid-soil tolerance of six strains (WU95, NA3001, WSM409, TA1, NA3025 and NA3039) of Rhizobium leguminosarum
bv. trifolii was assessed in a three-year cross-row ®eld experiment in an acid sandy soil of pH 4.2. Strains WSM409, NA3039
and WU95 were more acid-soil tolerant than strains NA3025, TA1 and NA3001. Strains WSM409 and NA3039 colonised and
persisted in acid-soil to a greater degree than strains TA1 and NA3001. The data from this study clearly identi®ed strain
WSM409 as a strain with outstanding potential for improving the production of clovers on acid soils. 7 2000 Elsevier Science
Ltd. All rights reserved.
Keywords: Soil acidity; Acid tolerance; Rhizobium leguminosarum bv. trifolii

1. Introduction
Agricultural systems have been developed in Australia based on the rotation of annual pasture legumes

with cereal crops using a range of management practices such as ley and phase farming strategies (Cocks
et al., 1980; Ewing et al., 1992; Reeve and Ewing,
1993). In these systems, the pasture legume is inoculated with an appropriate strain of root nodule bacteria during the year of establishment (Brockwell et al.,
1995). In subsequent years, the successful regeneration
of the legume pasture will depend on the survival of
the inoculant strain in the soil in the absence of the
legume host (Howieson, 1995a). In the Mediterranean
environment of southern Australia, this requires the
persistence of the root nodule bacteria in naturally

* Corresponding author. Tel.: +61-08-9360-2439; fax: +61-089360-6486.
E-mail address: ewatkin@central.murdoch.edu.au (E.L.J. Watkin).
1
Present address: Oce of the Pro Vice Chancellor (Research),
University Of Tasmania, Hobart, Tasmania, Australia.

acidic-soils over the hot, dry summer (Chatel and Parker, 1973a).
The failure of N2-®xing Rhizobium-legume symbioses
in acid soils is a signi®cant problem a€ecting agricultural production in many areas of the world (Coventry
and Evans, 1989; Wright and Zeto, 1991). Sustainable

production of legume pastures on acid soils can be
severely limited by poor growth and survival of root
nodule bacteria (Robson and Loneragan, 1970), which
die when exposed to acid conditions (O'Hara and
Glenn, 1994). Subterranean clover (Trifolium subterraneum ) is the most important and widely sown pasture
legume in southern Australia. Acid-tolerant strains of
clover root nodule bacteria would be bene®cial in
improving clover pasture production (Slattery et al.,
1992).
Studies on the e€ects of low pH on root nodule bacteria have focused largely on attempts to select acidtolerant strains for use as inoculants (Graham et al.,
1982; Thornton and Davey, 1983b; Howieson et al.,
1988). Much of the work has used growth on agar
plates (Brom®eld and Gareth Jones, 1980; Lindstrom
and Myllyniemi, 1987; Richardson and Simpson, 1989)

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
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E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403

and in broth culture (Thornton and Davey, 1983a;
Wood and Cooper, 1985; Richardson and Simpson,
1989) though there are some data from ®eld experiments (Thornon and Davey, 1984; Wood et al., 1984;
Lindstrom and Myllyniemi, 1987). Howieson and
Ewing, (1986) used a cross-row technique in a long
term ®eld experiment to study acid-soil tolerance in
Sinorhizobium spp. The cross-row procedure involves
sowing rows of inoculated seed in the ®rst year of the
experiment, and then, in subsequent years, sowing
uninoculated seed in rows at right angles to the original row. This technique assesses the persistence of
strains in the soil at the site of inoculation and also
determines their ability to colonise the soil away from
the point of initial inoculation.
The ability of inoculum strains of root nodule bacteria to colonise a soil in the absence of the host
legume is important in situations where it is advantageous to introduce the root nodule bacteria separate
from the host legume seed (Howieson, 1995b). These
situations may arise when fungicides are used on the
legume seed (Stovold and Evans, 1980; Evans et al.,

1986), or when the legume is sown in conditions that
are harmful to the survival of the bacteria. Therefore,
an understanding of the saprophytic competence of
inoculum strains is important in devising inoculation
strategies to overcome these problems (Howieson,
1995a).
In this study, we have used the cross-row technique
in a three-year ®eld experiment to examine the performance in an acid-soil (pH 4.2) of six selected strains
of Rhizobium leguminosarum bv. trifolii, hereafter
referred to as R. trifolii. Persistence and colonisation
was assessed over the three years of the experiment following the introduction of strains into the acid-soil
either as seed-coated peat inoculum, or as peat added
directly to the soil in the absence of the host plant.
2. Materials and methods
2.1. Experimental design
The experimental design was adapted from that of
Howieson and Ewing (1986) to evaluate acid tolerance

Fig. 1. Sowing and harvesting regimes. Plots sown in the ®rst year with (a) T. subterraneum inoculated with R. trifolii (±

±) or with (b) R. trifolii in the absence of a host (± ± ±). All plots sown with uninoculated T. subterraneum (ÐÐ) in the second and third years. Plants harvested in

the ®rst, second, and third years ( . . . . . .) 8 weeks after sowing.

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403

in strains of Sinorhizobium spp. (Fig. 1(a) and (b)). Six
strains of R. trifolii (Table 1), plus an uninoculated
control treatment, in factorial combination with host
Trifolium subterraneum (cv. Dalkeith), plus a nil host
treatment, in quadruplicate.
2.2. Site preparation
The experiment was carried out on the Dryland
Research Institute, Merredin, Western Australia (318
28 ' S, 1188 18 ' E). This area has a Mediterranean climate with an average annual rainfall of 315 mm. Seventy percent of the rain falls during the growing
season between May and October. The yearly rainfall
during the experiment was 405 mm in 1992, 266 mm
in 1993, and 154 mm in 1994. The site had been
cleared of the original vegetation of acacia and wodgil
in 1965, and cultivated in a cereal/voluntary pasture
rotation until 1982. Wheat/lupin rotation was sown
during 1983±1990; the lupin was inoculated with Bradyrhizobium sp. (Lupini ) strain WU425. During 1991

the site was under voluntary pasture. There is no
record of the site being sown to inoculated sub-clover.
The soil is a combination of Wodgil sand (Northcote:
Uc 5.22) and Ulva gravelly sand (Northcote: KS-Uc
4.21) with an organic carbon content of 0.9%, total
nitrogen of 0.055% and pH 4.2 (1:5 in 0.01 M CaCl2).
The site was sprayed two weeks prior to sowing the experiment with 1l/ha Roundup C.T. (active ingredient
450 g Glyphosphate/l) to kill weeds, then cultivated to
a depth of 2 cm.
2.3. Preparation of seed and inoculum
Rhizobial inocula were prepared using cultures of
the six strains of R. trifolii grown at 288C in Tryptone
Yeast (TY) broth (Beringer, 1974) to late exponential
phase (OD600 approx. 1.0). Twenty ®ve ml of culture
of each strain was aseptically injected into separate
bags of sterile peat (50 g, Biocare Technology, NSW,

Australia) to give a ®nal concentration of approx. 1 
109 cells gÿ1 peat. An uninoculated control peat was
prepared by injecting 25 ml of sterile TY broth into a

50 g bag of sterile peat. The inoculated and control
peats were thoroughly mixed by hand manipulation
and incubated at 288C for 10 days. The inocula were
then stored at 48C. Viable counts were performed on
the stored, inoculated peats one week before use in the
®eld experiment, and bags with similar numbers of rhizobia (2±5.5  108 cells gÿ1 peat) were used to inoculate sterile seeds.
Seeds of T. subterraneum were surface sterilised by
immersion (5 s) in 95% (v/v) ethanol followed by 3
min treatment with acidi®ed 0.01% (w/v) HgCl2 (Vincent, 1970). After six washes in sterile deionised water,
the seeds were air-dried aseptically in a laminar ¯ow
cabinet. Sterile dry seeds were immediately inoculated
and lime-pelleted by ®rst mixing 1 g of 2% (w/v) methocel adhesive solution with 0.5 g of the appropriate
peat, and then adding 50 g of the appropriate peat,
and then adding 50 g of clover seed and 35 g of ®nely
ground lime (Brockwell et al., 1982; Howieson and
Ewing, 1986). The seed was mixed aseptically until uniformly coated with peat and lime. Inoculated seed was
stored at 48C overnight and then sown.
2.4. Sowing
2.4.1. Plots sown with inoculated seed in year one
The experiment was sown on 8th June 1992 in ®ne

sunny conditions. Each treatment was sown in two
parallel rows, 2 m long and 0.5 m apart at a rate of
approximately 1 g of inoculated clover seed per meter
(Fig. 1(a)). Each plot was separated by a 1 m border
to minimise movement of inoculum strains between
plots. All plots were hand fertilised at the time of sowing with superphosphate equivalent to 200 kg haÿ1.
In the second and third years of the experiment,
regenerative growth was killed with Glyphosphate two

Table 1
Strains of Rhizobium leguminosarum bv. trifolii used in this study
Strain

Origin and characteristics

Source

NA3001

Acid media tolerant (Richardson and Simpson, 1989) isolated in New South

Wales. Minimum pH (agar) for growth, 4.3 (Watkin et al., 1997)
Isolated from acid-soil in the central tablelands of New South Wales. Minimum
pH (agar) for growth, 4.6 (Watkin et al., 1997)
Isolated from acid-soil in the central tablelands of New South Wales. Minimum
pH (agar) for growth, 4.6 (Watkin et al., 1997)
Commercial inoculum for white clover. Acid sensitive (Richardson and
Simpson, 1989). Minimum pH (agar) for growth, 4.6 (Watkin et al., 1997)
Isolated from T. subterraneum on acid-soil in Sardinia. Minimum pH (agar) for
growth, 4.5 (Watkin et al., 1997)
Commercial inoculum for T. subterraneum. Minimum pH (agar) for growth, 4.6
(Watkin et al., 1997)

G. Gemell, AIRCS

NA3025
NA3039
TA1
WSM409
WU95

1395

G. Gemell, AIRCS
G. Gemell, AIRCS
Murdoch University collection
JG Howieson Centre for Rhizobium Studies
Murdoch University collection

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E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403

weeks after the ®rst rain and again after a further
week. The plots were fertilised at the time of sowing
with superphosphate at a rate of 150 kg haÿ1.
In the second year, all undisturbed portions of the
original rows were resown with surface-sterilised clover
seeds on 4th June 1993. In addition, the right-hand
row of each plot had three 0.5 m rows sown at right
angles to, and intersecting to the right of the original

row (Fig. 1(a)).
In the third year, the plots were resown with surface-sterilised clover seeds on 23rd July 1994 in the top
1 m of the original left-hand row (Fig. 1(a)). In addition two 0.5 m rows were sown at right angles to,
and intersecting, to the left of the original left-hand
row. This was done to minimise contamination
between plots due to site disturbance in the second
year.
2.4.2. Plots inoculated without host in the ®rst year
In the ®rst year, those plots that were not to be
sown to clover had the peat inoculant of each strain
sprinkled along the two 2 m rows and covered with
soil (Fig. 1(b)). In the second year these rows were
sown to clover as described above. In the third year,
the total 2 m of the left-hand row was resown with
surface sterilised clover seed with four 0.5 m rows
sown at right angles and to the left of the original row
(Fig. 1(b)).
2.5. Harvesting
2.5.1. Plots sown with inoculated seed in ®rst year
In the ®rst year the plants in the lower 1 m of the
left-hand row of each plot were harvested 8 weeks
after sowing (Fig. 1(a)) to a depth sucient to recover
the total root system. The remaining plants in each
plot were left undisturbed.
In the second year, eight weeks after sowing, the
plants were harvested in the original right-hand row
and in the three cross rows (Fig. 1(a)) in the following
four sampling regions: (1). the original right-hand row,
(2). cross rows, 1±10 cm from the original row, (3).
cross rows, 11±20 cm from the original row, and (4).
cross rows, 21±50 cm from the original row.
In the third year, eight weeks after sowing, the
plants were harvested in the original top 1 m of the
left-hand row and the two cross rows (Fig. 1(a)), with
sampling regions as in the second year.
2.5.2. Plots inoculated without host plant
In the ®rst year there was no harvest (Fig. 1(b)). In
the second year the plants were harvested as described
above for plots sown with inoculated seed in the ®rst
year (Fig. 1(b)).
In the third year, the original left-hand row and two
cross rows were harvested in the four sampling regions

described above, except that the top and bottom 1 m
sections were harvested separately (Fig. 1(b)).
2.6. Assessment of nodulation, yield and nitrogen
At each harvest, all plants from each sampling
region within a plot (in total approx. 200 plants) were
pooled, 20 plants were selected randomly and then
assessed for nodulation using a scoring system adapted
from that of Brockwell et al. (1982). This scoring system ranks nodulation for size of nodules as well as location of nodules on the root system. The nodulation
scores for the 20 plants were averaged. Up to 25
nodules were picked from each of the 20 plants (i.e., to
a maximum of 500 nodules); these were stored at 48C
over granulated CaCO3 until the resident bacteria
could be typed.
The tops of the 20 plants were removed, bulked and
dried at 708C for 48 h and the dry weights measured.
Dry tops were milled ®nely and the total nitrogen was
determined using Kjeldahl digestion (Dalal et al.,
1984), and ammonia determined by the method of
Searle (1984).
2.7. Nodule typing
Nodule isolates were typed using pH sensitivity and
intrinsic antibiotic resistance patterns (Josey et al.,
1979), and con®rmed using polymerase chain reaction
with directed primers (Richardson et al., 1995).
Nodules were surface-sterilised by immersion in aqueous acidi®ed HgCl2 (0.01% w/v) for 0.5±2 min,
depending on nodule size. Sterilised nodules were then
thoroughly washed at least six times with sterile deionised water (Vincent, 1970). Each nodule was crushed
in a small drop of sterile deionised water with a sterile
orange stick and the suspension patched onto TY
agar. After 3±4 days incubation at 288C colonies were
respotted onto TY master plates and incubated for 3
days at 288C. The master plates were then replica plated onto minimal medium (Watkin et al., 1997) at pH
4.35, 4.45, 4.70 and 7.0, or TY agar containing chloramphenicol (20 mg mlÿ1), or kanamycin (20 mg mlÿ1),
or gentamycin (1.0 mg mlÿ1), or spectinomycin (20 mg
mlÿ1). A TY plate was included at the end of each run
to ensure adequate replication. Fifty nodules were
typed for each plot.
The identi®cation of the nodule isolates obtained
from the pH and antibiotic resistance pro®les were
con®rmed using polymerase chain reaction ampli®cation pro®les (Richardson et al., 1995). The primer
RPO1, a directed primer based on a reiterated Rhizobium nif promoter consensus element, was obtained
from BRESATEC and used following the protocols of
Richardson et al. (1995).
Strain NA3025, which forms large numbers of distinctive, extremely small (1 mm dia.) nodules, was

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403

identi®ed by nodule morphology in the the second and
the third years.
2.8. Dual nodule occupancy
For each plot, the exudates from ten sterilised
nodules were streaked onto TY agar and incubated at
288C for 3±4 days. Ten colonies from each nodule
were patched onto TY plates and incubated for a
further 3±4 days. These were then replica plated for
typing as described above.
2.9. Numbers of R. trifolii in the soil
The most probable number (MPN) of indigenous
strains of R. trifolii at the ®eld site prior to sowing in
1992, and the population level of the six inoculant
strains of R. trifolii in the ®eld plot at the time of sowing in the second and third years, were determined
using the plant infection test of Brockwell (1982)
except screwcap tissue culture tubes (11  4 cm dia.)
were used instead of test tubes. In the second year, rhizobial numbers in the soil at the point of original inoculation were estimated at depths 0±2 cm and 2±5
cm. In the third year, the 0±5 cm pro®les were bulked.
The minimum level of detection for this assay is 11
rhizobia gÿ1 soil with 95% con®dence limits (Woomer
et al., 1988).
2.10. Presence of background R. trifolii
Samples of the top 5 cm of soil were collected
during April 1992, prior to the break of season from
three locations at the ®eld site and stored for 2 days
prior to mixing, air drying and sieving. The soil was
then placed into 3 kg pots and watered to ®eld capacity (13.5% w/v) with sterile, deionised water. Surface sterilised seeds of T. subterraneum cv. Dalkeith
were imbibed in sterile, deionised water for 4 h and
germinated on water agar at 258C for 48 h in the dark.
Six germinated seeds were sown at a depth of 1 cm in
each pot, covered with soil and a 1 cm layer of sterile
vermiculite. The pots were maintained in a root cooling tank at 21218C and watered to ®eld capacity with
sterile water through watering tubes on every second
day. The plants were harvested at 6 weeks and roots
examined for the presence of nodules.
3. Results and discussion
3.1. Background population of R. trifolii
No naturalised strains of R. trifolii were detected in
the soil samples collected from the ®eld site prior to
sowing the experiment in 1992 i.e. nodules did not
form on the roots of any of the plants used for the
MPN estimation. In addition, none of the plants

1397

grown in potted soil collected from the site were nodulated after six weeks growth in the glasshouse.
In the ®rst year of the experiment, 5% of clover
plants grown in the uninoculated plots were ine€ectively nodulated by naturalised strains of R. trifolii.
This nodulation consisted of one or two white (ine€ective), large nodules being present on the extreme lateral roots. Using intrinsic antibiotic resistance and
growth on low pH agar plates, ®ve di€erent strains
were identi®ed from these isolates. Plants sown in the
second and third years in uninoculated soil, not previously sown with clover, were not nodulated by naturalised strains.
Naturalised populations of R. trifolii are present in
many soils in the agricultural regions of south-west
Australia (Parker, 1962; Chatel and Parker, 1973b).
Population density, e€ectiveness, and competitive ability have been identi®ed as the primary characteristics
of indigenous rhizobial populations that a€ect inoculation responses (Thies et al., 1991). Naturalised populations of root nodule bacteria can signi®cantly limit
responses to inoculation as long as the population contains some e€ective strains (Singleton and Tavares,
1986).
The background population of R. trifolii in the soil
at the site of this study comprised ®ve ine€ective
strains with very poor competitive ability. These
strains were only detected in the uninoculated control
plants grown during the ®rst season of the experiment
and were not detected in the subsequent seasons. Perhaps the dry, below average rainfall in 1993 and 1994
reduced their limited capacity to nodulate the uninoculated clover. Clearly the background populations of
naturalised strains of R. trifolii were not a signi®cant
factor in the performance of the inoculum strains in
this experiment. From this data, it would seem reasonable to propose that there was little competition for
inoculum strains from the naturalised strains and as a
consequence the inoculant strains were able to easily
nodulate the clover in the ®rst year of the experiment.
3.2. Movement of inoculum strains between plots
In the second year of the experiment, there was proli®c nodulation of sub-clover plants grown in plots
which had previously been sown to an uninoculated
host in the ®rst year (Table 2, Nil treatment). Typing
of the isolates from these nodules consistently showed
that this nodulation was entirely due to the presence in
the uninoculated plots of the inoculant strains from
immediately adjacent plots. The majority of isolates
from nodules in uninoculated plots were the strain
that had been inoculated into the adjacent plot on the
east side. This indicates that a consistent pattern of
movement occurred mostly in an east to west direction, possibly caused by the strong easterly winds in

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E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403

Table 2
The percentage of plants nodulated and nodule score in the original row in the second and third year for the six strains of Rhizobium leguminosarum bv trifolii and the uninoculated controla
Inoculant strain

NA3001
NA3025
NA3039
TA1
WSM409
WU95
Nil

Second year

Third year

Percentage of plants nodulated

Nodule score

Percentage of plants nodulated

Nodule score

78.8a (22.1)
87.5ab (18.9)
98.8b (2.5)
86.7ab (5.8)
97.5b (5.0)
78.8a (18.9)
96.3b (4.8)

3.7b (1.4)
6.6a (3.6)
5.8a (1.4)
2.3c (2.1)
5.6a (1.3)
5.8a (1.3)
5.9a (0.3)

71.7abc (27.9)
72.0abc (30.4)
77.5ab (33.0)
58.8c (33.3)
70.6abc (24.0)
86.7a (12.6)
53.3c (18.9)

4.5ab (0.6)
5.2b (1.7)
5.8b (1.6)
3.5a (1.4)
4.7ab (0.5)
4.5ab (0.7)
4.5ab (0.7)

a

Values are the means of four measurements with standard deviations in parenthesis. Values in each column followed by the same letter are
not signi®cantly di€erent (P = 0.05). Analyses of variance were performed on the arcsin transformation of percentage of plants nodulated raw
data and on the square root transformation of nodule score raw data.

summer moving dry soil from plot to plot. The fact
that there was no evidence for movement of strains to
plots not previously sown with sub-clover (Table 3,
Nil treatment) may indicate that the presence of senescent plant material above ground may trap the dry
dust and enhance colonisation of the soil. In addition,
presence of senescing roots of sub-clover may have
promoted the establishment of the rhizobia in these
plots.
3.3. Persistence of inoculant strains in the presence of
the host plant
In the ®rst year of the experiment, all inoculated
plants had abundant nodules on their crown roots.
Typing of the isolates from these nodules using intrinsic antibiotic resistance and pH sensitivity pro®les
showed that all the nodules produced in an inoculated
plot were formed by the inoculum strain (data not
shown). The persistence of the inoculant strains in the

soil at the site of inoculation was assessed over a three
year period in two ways. Firstly, by MPN counts of
populations of inoculum strains in soil at the point of
original inoculation collected at the time of sowing in
second and third years. Secondly, by examining the
nodulation of plants sown in the second and third
years in the original inoculated row. The percentage of
plants nodulated by inoculant strains in years subsequent to initial inoculation and sowing is generally
considered a measure of persistence at the site of inoculation (Chatel and Greenwood, 1973; Howieson,
1995a).
The MPN data show that at the time of sowing in
the second year of the experiment strains NA3039,
NA3025 and WSM409 were present in the soil at more
than 103 cells/g of soil in the top 0±2 cm and at a
depth of 2±5 cm. Strain TA1 was present at 102 cells/g
of soil in the surface horizon and could not be
detected at 2±5 cm depth. Strain NA3001 was just
detectable in the top 0±2 cm of soil at 11 cells/g of soil

Table 3
The percentage of plants nodulated and nodule score in the original row in the second and third year for the six strains of Rhizobium leguminosarum bv trifolii inoculated into the soil in the ®rst year in the absence of a host and the uninoculated controla
Inoculant strain

NA3001
NA3025
NA3039
TA1
WSM409
WU95
Nil

Second year

Third year

Percentage of plants nodulated

Nodule score

Percentage of plants nodulated

Nodule score

25.0a (28.6)
60.0c (39.4)
91.3b (17.5)
33.8a (37.7)
92.5b (11.9)
95.0b (10.0)
1.3d (2.5)

1.3a (1.1)
8.0c (4.8)
5.0b (1.8)
1.9a (2.6)
4.8b (1.0)
6.1bc (2.6)
1.0a (1.0)

15.8c (13.6)
63.7b (24.4)
80.8ab (20.1)
33.6c (32.1)
84.7a (11.7)
77.5ab (27.2)
9.1c (12.9)

2.3ab (1.7)
3.4ab (0.4)
4.4bc (0.5)
2.7ab (1.4)
3.5ab (1.4)
5.8c (0.8)
4.0abc (4.0)

a
Values are the means of four measurements with standard deviations in parenthesis. Values in each column followed by the same letter are
not signi®cantly di€erent (P = 0.05). Analyses of variance were performed on the arcsin transformation of percentage of plants nodulated raw
data and on the squareroot transformation of nodule score raw data.

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403

but was present at 2  104 cells/g soil at 2±5 cm. Strain
WU95 was not detected in soil from plots inoculated
with this strain in the ®rst year. There were clear
di€erences between the strains in their capacity to
maintain populations in the soil during the long, dry
summers.
WSM409 and NA3025 were the only strains present
in detectable numbers in soil collected at the time of
sowing in the third year of the experiment at 26 and
11 cells/g soil. The third year of the experiment was
delayed by a late break of season and sown six weeks
later in the year than in the two previous seasons. The
surviving inoculant bacteria were, therefore, subjected
to a longer period of desiccation stress in the third
year and this may have been a signi®cant factor reducing the populations of inoculant strains present in the
soil. It might be anticipated that low populations of
surviving inoculant may reduce early e€ective nodulation of regenerating clover under these conditions. The
detectable populations of WSM409 and NA3025 indicate that these strains were present in greater numbers
in the soil after a period of considerable desiccation
and the acid-soil tolerance of these two strains. R. trifolii was not detected in uninoculated soil collected at
the time of sowing in the second and third years of the
experiment.
Typing of isolates from nodules, formed on plants
sown in inoculated plots, showed that the nodulation
of these plants in the second year was almost entirely
due to the inoculant strain. The percentage occupancy
of nodules from inoculated plots due to the inoculant
strain ranged from 74% for strain NA3001 to 97% for
strain WU95. Low levels of dual nodule occupancy
were observed in the second year of the experiment
ranging from 1.3% for strain NA3001 to 6.7% for
strain NA3039. The co-occupant strain was not identi®ed in these cases. Thus, the nodules formed on plants
sown in the second year in plots inoculated in the ®rst
year were predominantly formed by the strain added
to the soil in the ®rst year. The inoculum strains dominated the nodulation of clover plants in inoculated
plots in all three years of the experiment.
In the second year, the plots inoculated with strains
WSM409 and NA3039 had a signi®cantly higher percentage of plants nodulated in the original row than
those plots inoculated with strains WU95 and NA3001
(Table 2). The lower nodule scores for strains TA1
and NA3001 in the second year (Table 2) were a result
of nodules only being formed by these two strains on
the lateral roots. Strains WSM409, WU95, NA3025
and NA3039 all produced high nodule scores (Table 2)
as a consequence of the nodules being positioned on
the crown and upper lateral roots.
In the third year, strain WU95 nodulated a signi®cantly higher proportion of plants in the original
inoculated row than TA1 (Table 2). Strains NA3039

1399

and NA3025 had higher nodule scores than strain TA1
(Table 2).
Strains WSM409 and NA3039 performed as well as,
or better than, the commercial inoculant strain WU95
in the plots sown each year to sub-clover. These three
strains clearly have the capacity to stongly colonise the
acid-soil at the site of inoculation and persist from
year to year in long term pastures. The poorer performance of strains NA3001, and TA1 in persisting at
the site of inoculation indicates that these two strains
may be impaired in their capacity to colonise this acidsoil from the site of inoculation. For TA1, this may indicate acid-soil sensitivity and for NA3001 this may be
either a consequence of its ine€ectiveness or an indication of acid-soil sensitivity in this strain that is tolerant of acidity in the laboratory.
3.4. Persistence of inoculant strains without the host
plant
The survival of strains in the acid-soil in the absence
of their host plant was examined by inoculating plots
in the ®rst year with peat inoculum alone. Sterilised
clover seeds were subsequently sown in the inoculated
rows in the second and third years, and the nodulation
of these plants was used to assess the persistence of
the inoculum. In this situation, strains WSM409,
WU95 and NA3039 nodulated a signi®cantly higher
percentage of plants in the second year at the site of
inoculation than the other strains (Table 3); they had
also signi®cantly greater nodule scores than strains
NA3001 and TA1 in the second year (Table 3). In
these plots, strains WSM409, WU95 and NA3039
formed medium sized nodules (2±5 mm dia.) on both
crown and lateral roots (early nodulation possibly indicative of a larger population of these strains). In
contrast, strains NA3001 and TA1 produced nodules
only on lateral roots (late nodulation indicative of a
small population). The high nodule scores recorded for
strain NA3025 were due to the formation of numerous
very small, white nodules (1 mm dia.) on the crown
region of the roots.
In the third year, after the absence of a host plant in
the ®rst year and the presence of clover in the second
year, strains TA1 and NA3001 produced a signi®cantly
lower percentage of nodulated plants (Table 3). Strains
WU95 and NA3039, produced crown nodules, whereas
the other strains only formed nodules on lateral roots.
The six strains R. trifolii di€ered in their persistence
in the acid-soil for two years without clover being present. In this situation, the level of persistence shown
by each strain was similar to that shown by the strain
when the host plant was absent for only one year.
Strains WSM409, WU95, NA3039, and NA3025 nodulated a signi®cantly greater percentage of plants than
strains NA3001 and TA1 (Table 4). Strains WU95,

1400

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403

WSM409, and NA3025 produced crown nodules and
strains NA3001, NA3039, and TA1 formed nodules
only on the lateral roots. Strain WU95 had a signi®cantly larger nodule score than strains NA3001,
NA3039, and TA1 (Table 4).
Previously, Gemell and Roughley (1993) found that
strain NA3001 persisted poorly in an acid-soil in
NSW, and they attributed the poor survival of this
strain to competition from indigenous strains. In the
study reported here, the poor performance of strain
NA3001 was clearly not related to strong competition
from the background strains in the soil at the time of
establishing this experiment. In addition, the presence
of other inoculant strains in plots in the second and
third years of the experiment did not provide strong
competition for strain NA3001 because 75±85% of
nodules formed on plants sown in plots inoculated
with NA3001 contained this strain. Thus, the poor performance of strain NA3001 in this Western Australian
soil was more likely to have been due to its poor survival. The same strain has been reported to persist in an
acid-soil in Victoria (Slattery pers. com.), indicating
that di€erences in background rhizobial populations
and soil characteristics between sites can a€ect strain
survival.
3.5. Colonisation of acid-soil by R. trifolii
Colonisation of soils by the inoculum strain of root
nodule bacteria is essential for the establishment of
productive pastures because of the need for e€ective
nodulation of plants located at points distant from the
site of inoculation. The colonisation of the acid-soil by
the six inoculant strains was assessed in the second
and third years of the experiment by observing the
nodulation of clover plants sown in cross rows at distances from the original inoculated row. There were
considerable di€erences between strains in their ability
to colonise the acid-soil and nodulate plants at a distance from the original site of inoculation. Strains

WSM409, NA3039, and WU95 were the most successful strains in colonising the acid-soil and strains TA1
and NA3001 were the poorest colonisers. In the second
year of the experiment, strains NA3039 and WU95
nodulated a signi®cantly greater percentage of plants
than strains NA3001, NA3025 and TA1 in the zones
at distances of 1±10 cm and 11±20 cm from the original inoculated row (Table 5). At 21±50 cm, strain
NA3039 nodulated a signi®cantly higher percentage of
plants than strains TA1, NA3001, and NA3025
(Table 5). Strains WU95, NA3039, and WSM409 produced signi®cantly higher numbers of nodules at 21-50
cm than strains TA1, NA3001 and NA3025 (Table 5).
Plants were poorly nodulated when sown in cross rows
at distances greater than 11 cm from the original
inoculated row on plots inoculated with strains TA1,
NA3001 and NA3025 (Table 5).
The results from the third year of the experiment
were compromised by an extremely dry year with only
154 mm of rainfall compared to an average annual
rainfall of 315 mm. This resulted in very poor establishment and survival of clover sown in this season. As
a consequence, statistical analyses could not be performed on results because of the missing data. However, observations of the pattern of nodulation in the
cross rows provided evidence for the survival of strains
in this extremely dry season. All strains except
NA3025, were able to nodulate plants at 1±10 cm
(Fig. 2); strains NA3001, WSM409, and NA3039
nodulated plants at 11±20 cm (data not shown) and
strain WSM409 was the only strain to nodulate plants
sown at a distance of 21±50 cm from the original
inoculated row. This strongly indicates that this strain
was the best at colonising the acid-soil at a substantial
distance from the point of inoculation. The success of
WSM409 in the third year of the experiment may have
also been a result of the ability of this strain to rapidly
nodulate young roots. Early nodulation results in
more productive growth of roots and may have

Table 4
The percentage of plants nodulated and nodule score in the third year in the original row for the six strains of Rhizobium leguminosarum bv. trifolii and the uninoculated control inoculated into the soil in the ®rst year in the absence of a host. Host also absent in the second yeara
Strains

Percentage of plants nodulated

Nodule score

Number of replicates

NA3001
NA3025
NA3039
TA1
WSM409
WU95
Nil

28.7b (33.6)
76.0a (25.7)
69.3a (21.8)
43.9b (26.4)
78.4a (32.6)
77.4a (21.8)
NG

3.1a (1.9)
4.3ab (1.5)
4.1a (1.0)
2.9a (2.6)
4.5ab (0.4)
5.9b (2.0)
NG

4
3
3
3
4
3

a

Values are the means of four measurements with standard deviations in parentheses. NG = plants did not grow in any of the four replicates.
Values in each column followed by the same letter are not signi®cantly di€erent (P = 0.05). Analyses of variance were performed on the arcsin
transformation of percentage of plants nodulated raw data and on the squareroot transformation of nodule score raw data.

1401

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403

Table 5
The percentage of plants nodulated and nodule score at 1±10, 11±20 and 21±50 cm from the original row in the second year for the six strains of
Rhizobium leguminosarum bv trifolii and the uninoculated controla
Inoculant strain

NA3001
NA3025
NA3039
TA1
WSM409
WU95
Nil

Percentage of plants nodulated

Nodule score

1±10 cm

11±20 cm

21±50 cm

1±10 cm

11±20 cm

21±50 cm

53.8a (28.1)
70.0ab (28.3)
88.8c (9.5)
63.3ab (17.6)
77.5bc (11.9)
93.8c (7.5)
58.8a (16.0)

36.3a (39.3)
41.2ab (27.8)
71.2c (24.6)
33.3a (10.4)
57.5bc (15.0)
72.5c (18.5)
41.3ab (11.1)

25.0ab (26.5)
26.3ab (42.7)
55.0c (37.4)
20.0a (13.2)
38.8bc (23.2)
41.3bc (19.3)
17.5a (14.4)

3.6ab (1.3)
2.7b (1.8)
4.3a (1.8)
3.1ab (0.9)
4.0ab (1.2)
4.4a (1.3)
3.0ab (0.4)

1.6b (1.4)
3.1a (1.3)
3.8a (2.3)
1.1b (0.6)
3.7a (1.0)
3.8a (1.4)
3.6a (0.9)

1.9b (1.3)
1.5b (1.2)
3.5a (2.1)
0.7b (0.2)
3.1a (1.0)
3.1a (1.6)
1.4b (0.5)

a

Values are the means of four measurements with standard deviations in parenthesis. Values in each column followed by the same letter are
not signi®cantly di€erent (P = 0.05). Analyses of variance were performed on the arcsin transformation of percentage of plants nodulated raw
data and on the square root transformation of nodule score raw data.

assisted the survival of plants during the water stress
that occurred later in the third year.
3.6. Colonisation of the soil in the absence of a host
A more extreme test of the ability of R. trifolii to
survive and colonise the acid-soil was the situation
where plots were inoculated in the ®rst year in the
absence of the host and then sown with sterilised clover in the second year. Plots containing strains WU95,
WSM409, and NA3039 had signi®cantly more plants
nodulated at 1±10 cm than strains TA1, NA3001 and
NA3025 (Table 6). Plants nodulated by NA3039 had a
signi®cantly higher nodule score than NA3001,
NA3025, TA1, and WSM409, (Table 6), with nodules
formed on the crown and lateral roots. Strains TA1
and NA3001 had signi®cantly lower nodule scores
Table 6
The percentage of plants nodulated and the nodule score at 1±10,
and 11±20 cm from the original row in the second year for the six
strains of Rhizobium leguminosarum bv trifolii and the uninoculated
control inoculated into the soil in the ®rst year in the absence of a
hosta
Inoculant

Percentage of plants nodulated

Nodule score

strain

1±10 cm

11±20 cm

1±10 cm

11±20 cm

NA3001
NA3025
NA3039
TA1
WSM409
WU95
Nil

33.8bc (35.0)
40.0c (31.1)
75.0d (28.0)
16.3ab (2.5)
61.3d (43.3)
62.5d (35.2)
6.25a (7.5)

13.8ab (24.3)
11.3ab (13.2)
33.3d (30.8)
21.3abc (11.9)
30.0cd (14.7)
25.5bcd (15.4)
6.25a (6.3)

0.9a (0.8)
3.1b (2.2)
5.5c (2.0)
1.4a (0.8)
3.2b (2.8)
3.9bc (1.5)
3.0b (0.4)

0.9a (1.2)
4.3cde (3.4)
1.8bc (1.5)
1.3ab (1.1)
2.9cde (1.6)
2.4bcd (1.4)
3.6cde (0.9)

a
Values are the means of four measurements with standard deviations in parenthesis. Values in each column followed by the same
letter are not signi®cantly di€erent (P = 0.05). Analyses of variance
were performed on the arcsin transformation of percentage of plants
nodulated raw data and on the squareroot transformation of nodule
score raw data.

than the other strains (Table 6) with nodules only
forming on lateral roots. In the 11±20 cm region strain
NA3039 had signi®cantly better nodulation than
strains NA3001, NA3025, and TA1 (Table 6) and
strains TA1 and NA3001 had the lowest nodule scores
(Table 6).
The data from the third year of the experiment
could not be statistically analysed because the poor
survival of plants resulted in large amounts of missing
data. However, some plants did survive and nodulate
in the cross rows at a distance from the original row.
In plots where the host was absent in the ®rst year,
only strains WSM409, WU95, NA3025, and NA3039
nodulated 30% or more of plants in the 1±10 cm
sampling region (Fig. 2). In the plots, where the host
was absent in the second and third years strains
NA3039, WSM409, and WU95 produced more than
50% plant nodulation at 1±10 cm (Fig. 2), whilst in
the 21±50 cm region only strains WSM409 and
NA3039 produced any nodulation at all (data not
shown).
3.7. E€ectiveness of inoculant strains
As a measure of e€ectiveness of nitrogen ®xation,
total nitrogen of the clover plants at the ®rst year harvest, was expressed as percent nitrogen and mg nitrogen per plant (Table 7). Clover plants inoculated with
WU95, WSM409, and TA1 produced similar nitrogen
concentrations. However, when expressed as nitrogen
accumulation per plant, those plants inoculated with
WSM409 had the highest amount followed by WU95
and then plants inoculated with TA1. Plants inoculated with NA3025 contained a lower percentage of
nitrogen and lower amount of nitrogen per plant than
the uninoculated controls (Table 7).
Acid-soil-tolerant and acid-soil sensitive strains of R.
trifolii have been identi®ed using a ®eld experiment
based on the cross-row technique to determine the

1402

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403

Fig. 2. Percentage of plants nodulated at 1±10 cm from the original row in the third year for six strains of Rhizobium leguminosarum bv. trifolii
and the uninoculated control. Values are the means of 1±4 datum points.

ability of the strains to persist and colonise the soil.
Strains WSM409 and NA3039 were better colonisers
of the acid-soil (pH 4.2) than the commercial inoculum
strain WU95, and the two acid tolerant strains were
more e€ective in the ®eld than WU95. The data from
this study and from work determining the broad host
range of WSM409 (Howieson et al., 2000) provides
clear support for the selection of WSM409 as an acidsoil tolerant inoculum strain for a range of Trifolium
spp. to replace WU95. The use of WSM409 should
Table 7
The e€ectiveness of the inoculant strains of Rhizobium leguminosarum bv. trifolii for nitrogen ®xation in the ®rst year as expressed
by percent nitrogen and nitrogen harvested per plant (mg) of 20
bulked plants harvested from the original rowa
Inoculant strain

Percent nitrogen

Nitrogen per plant (mg)

NA3001
NA3025
NA3039
TA1
WSM409
WU95
Nil

4.05bc (0.25)
3.08a (0.76)
4.28c (0.22)
4.75d (0.06)
4.81d (0.33)
4.84d (0.25)
3.82b (0.83)

4.98b (0.46)
3.85a (0.70)
6.61c (1.61)
7.93d (1.56)
11.33f (2.40)
8.85e (8.10)
6.08c (2.01)

a
Values are means of 4 measurements with standard deviations in
parenthesis. Values in each column followed by the same letter are
not signi®cantly di€erent (P = 0.05).

increase the productivity of clover-based pastures on
acidic-sandy soils in southern Australia. The characterisation described here of acid-soil sensitive and acid
soil-tolerant strains of R. trifolii provides resources for
laboratory-based investigations on the basis of acidsoil tolerance in R. trifolii.
Acknowledgements
We gratefully acknowledge research support from
the Grains Research and Development Corporation.
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